A surface plasmon wave (SPW) is a charge density (electromagnetic) wave, excited at the interface between a conductor or semiconductor, e.g. a metal surface, and a dielectric. (N. Marschall, B. Fischer, "Dispersion of surface polaritons in GaP," Physical Review Letters 28, pp. 811-813, March (1972). Surface plasmon waves (SPWs) can be optically excited by an evanescent field, created when light undergoes total internal reflection, for instance, off the base of a prism (Kretschmann, E. (1971), "Die bestimmung optischer konstanten von metallen durch anregung von oberflachenplasmaschwingugen," Z. Phys. 241:313), or as it propagates down a fiber optic. The evanescent field penetrates through the metal and excites an SPW on the outer surface of the metal where the metal meets the dielectric. In all SPR configurations, SPR can be excited only by transverse magnetic (TM) polarized light, that is, light which has its electric field polarized parallel to the incident plane. (If transverse electric (TE) light is present in the system, it does not carry any SPR signal. It could possibly be used as a reference signal.)
The SPW is a resonant phenomenon which depends on the indices of refraction and the thickness of the various layers, as well as the wavelength and the angle of incidence of the light. The dispersion equation for an SPW is given by Equation 1: ##EQU1## where k.sub.o is the free space wavevector (k.sub.o =.omega./c); .di-elect cons..sub.c and .di-elect cons..sub.d are the complex permittivities of the conducting and the dielectric layers, respectively; and .omega. is the angular frequency. In order to excite the SPW, the parallel component of the incident wavevector k.sub..parallel., must equal the surface plasmon wavevector k.sub.sp. The parallel component of the incident wavevector can be expressed as Equation 2: ##EQU2## where n is the index of refraction of the medium in which the light is incident, .theta. and .lambda. are the angle of incidence and wavelength of the light, respectively.
It can be seen from Eq. (2) that the resonance condition can be met by either varying the angle or the wavelength of the incident light, until k.sub.sp =k.sub..parallel.. These techniques are referred to as angle modulation and wavelength modulation, respectively.
The SPW is highly localized at the surface of the conducting layer, e.g. a metal film. The intensity of the electric field of the SPW decays exponentially into the solution surrounding the dielectric. The characteristic decay length is given by (Raether, H. (1977), Physics of Thin Films, Academic Press, New York, p. 164) Equation 3: ##EQU3## where L represents the distance at which the intensity of the SPW has decayed to e.sup.-1 times its value at the conductor/dielectric interface. The wavevector k.sub.s =k.sub.o n.sub.s is the wavevector in the solution surrounding the sensor. Re refers to the real part of the quantity in paratheses. For example, if the surrounding medium is water and the conductor is gold, L=831 .ANG. at a wavelength of 6328 .ANG.. This makes it possible to determine very small changes in the effective index of refraction within approximately 2000 .ANG. of the gold surface. For example, surface plasmon resonance (SPR) sensors have been used to detect monolayer growth of Langmuir-Blodgett films (Mar, M. et al. (1993), "In-situ characterization of multilayered Langmuir-Blodgett films using a surface plasmon resonance fiber optic sensor," Proc. of the 15th Annual Conf. of the IEEE Engineering in Medicine and Biology Soc., San Diego, Calif, pp. 1551-1552).
Sensors based on the surface plasmon resonance (SPR) effect sense the refractive index (RI) of a thin region adjacent to the sensing surface (Sambles, J. R. et al. (1991), "Optical excitation of surface plasmons: an introduction," Contemporary Physics 32:173-183). SPR can be applied indirectly to other sensing applications by treating or manipulating the sensing surface such that the refractive index at the surface varies with the presence of the substance to be sensed. For instance, the surface can be made sensitive to a particular antibody by coating the surface with an antigen for that antibody. When the antigen binds to the antibody, the refractive index at the surface changes slightly. A commercial application of SPR to biological sensing has been developed using this principle (Liedberg, B. et al. (1995), "Biosensing with surface plasmon resonance--how it all started," Biosensors and Bioelectronics 10:i-ix). Other applications as diverse as sensing of ammonia (Van Gent, J. et al. (1991), "Realization of a Surface Plasmon Resonance-based Chemo-Optical Sensor," Sensors and Actuators A 25-27:449-452), magnetic and electric fields (Moslehi, B. et al. (1991), "Optical magnetic and electric field sensors based on surface plasmon polariton resonant coupling," Electronics Letters 27:951-953), and temperature (Chadwick, B. and Gal, M. (1993), "An optical temperature sensor using surface plasmons," Jpn. J. Appl. Phys. 32:2716-2717) similarly rely on sensitive overlayers.
The practical effect of a change in the RI of the dielectric adjacent to the SPR sensing surface is a shift in the SPR resonance curve. If the wavelength modulation technique is being used, the resonance curve of interest is the reflected intensity of light versus the incident wavelength. The minimum of this curve is defined as .lambda..sub.sp, which is the SPR resonance minimum in wavelength space. If the angle modulation technique is being used, the resonance curve of interest is the reflected intensity of light versus the incident angle. The minimum of this curve is defined as .theta..sub.sp, which is the SPR resonance minimum in angle space. It is also possible to determine the resonance from looking at the transmitted light intensity using either of these techniques. The techniques can also be combined, in which case the three dimensional intensity-angle-wavelength space must be considered. The absorption of the dielectric layers, which is directly related to the imaginary part of the refractive index of the dielectric layers, can also be determined from the SPR resonance. More absorbing dielectric layers, such as dye indicators (for instance, methylene blue), cause broader, less deep resonances. Parameters such as the resonance depth .delta..sub.sp, or the resonance width W.sub.sp, are not used as much as the resonance minimum location given by .lambda..sub.sp or the resonance angle given by .theta..sub.sp, because SPR is much more sensitive to changes in the real part of the index of refraction than it is to changes in absorption (see C. C. Jung, "Surface plasmon resonance," Masters Thesis, University of Washington (1991), C. Jung, R. Jorgenson, C. Morgan, and S. Yee, "Fiber optic surface plasmon dispersive index sensor for highly opaque samples," Process Control and Quality, 7, pp. 167-171 (1995).
In general, SPR sensors have been commonly used in two main applications: 1) measuring the refractive index of chemical samples and 2) monitoring the growth of thin biochemical or inorganic films on the surface of the sensor.
In general, an SPR configuration includes a source of electromagnetic radiation (light), an optically transmissive (transparent) component (the SPR sensor) which has a conducting film (e.g. a metal layer) on it, and a detector. The conducting film is in contact with a dielectric. Light is transmitted into the transparent component, undergoes total internal reflection, and if the conditions outlined in the equations above are met, then a surface plasmon wave will occur at the surface of the conducting film, that is at the interface of the metal layer and the dielectric. The detector measures the resonant phenomenon.
Surface plasmon resonance (SPR) can be performed in many different configurations. U.S. Pat. No. 4,844,613 discloses an SPR sensor configuration wherein the sensor includes a prism, one surface of which is coated with index matching fluid which is then covered with a glass microscope slide or cover slip. The exposed surface of the slide is then coated with a conductive layer (metal) on which SPR occurs. The metal layer can be coated with a sensitive layer, e.g. an antibody. The sensor is exposed to a test solution. If the antibody binds an antigen in the test solution, the thickness of the sensitive layer is changed, causing a change in the refractive index and a corresponding change in the SPR resonance angle.
U.S. Pat. No. 5,035,863 discloses an SPR sensor configuration adapted for biochemical testing on large samples, such as electrophoresis gels, allowing for sequencing of the gel based on changes in refractive index. The sensor includes several layers: a transparent plate, a metal layer of a mosaic of silver dots, and sandwiched in between is the gel. Light is directed and undergoes total internal reflection at the interface of the transparent plate and the metal layer and SPR occurs at the metal layer.
U.S. Pat. No. 4,997,278 discloses an SPR sensor configuration to detect the progression of a reaction between a sample and a sensitive layer, e.g. an antibody layer. An optically transmissive component comprises a hemicylindrical lens and slide. The slide is coated with a reflective layer (metallic film). A sensitive layer, the refractive index of which changes as the reaction, e.g. antigen binding to antibody, is applied to the surface of the reflective layer. The sensitive layer is smaller than the reflective layer. Collimated light is introduced via a lens which focuses the incident light to a point to form a fan-shaped area of light incident at the point. The light undergoes total internal reflection at the point and exits from the component. A detector array monitors the angle of incidence of the light at the point of SPR, together with a range of angles about it, to detect the progression of the resonant phenomenon, an indicator for the reaction between the sensitive layer (e.g. antibodies) and the sample (e.g. antigen).
U.S. Pat. No. 5,064,619 discloses another SPR sensor configuration which can be used to monitor the progress of a reaction, e.g. the binding of a sample to a sensitive layer (for instance, the binding of an antigen to an antibody). A laser is used to generate light which is reflected off a concave reflector to a point or line on the interface between a metal layer and a glass slide. A single beam of laser light is scanned by a movable mirror across an arc to cover the angles of incidence within which SPR occurs. The sensitive layer is coated on top of the metal layer, and a sample is passed across the sensitive layer. Any reaction (binding of antigen to antibody, for instance) causes a change in the refractive index of the layer. This change is detected by measuring the strength of the beam reflected from the point or line of incidence.
U.S. Pat. No. 5,055,265 discloses an SPR sensor configuration utilizing long-range SPR to follow the progress of a reaction, again between a sensitive layer and a test sample. This configuration includes a source of light, and a sensor which includes a block of transparent material, a dielectric membrane layered on the block, which membrane is coated with a layer of metal (only about 17 nm thick, versus about 56 nm for short range SPR). On top of the metal layer is a sensitive layer, e.g. antibody layer. Again, a sample to be tested is contacted with the combined layers (metal and sensitive) to test for the presence of a specific material. A radiation source and detector complete the configuration, allowing for measurement of the angle of incidence. Long-range SPR provides increased sensitivity.
U.S. Pat. No. 5,478,755 provides another SPR sensor configuration utilizing long-range SPR to monitor the progress of a reaction between a sensitive layer and a test sample (an immunoassay).
U.S. Pat. No. 5,047,213 discloses an SPR sensor configuration for biological sensing. This sensor uses an optical waveguide, e.g. a fiber optic. Electromagnetic radiation is introduced into the input end of the waveguide. The output end of the waveguide is cut off at an angle at its axis so that it has a sloping face, which is coated with a layer of metal, which itself is coated with a sensitive layer. The light undergoes total internal reflection at this face. A sample to be tested is contacted with the multi-layer. The light incident on the face leads to SPR, and the reaction of the sample with the sensitive layer can be monitored thereby.
U.S. Pat. No. 5,313,264 discloses another SPR sensor configuration for biological sensing. The configuration includes a sensor unit with at least two sensing surfaces, a source of light, and a lens for focusing the light into a wedge-shaped streak of light which extends transversely over all the sensing surfaces. A two-dimensional matrix of individual photodetectors provides the detecting device. An anamorphic lens system images rays of reflected light from the sensing surfaces onto each of its columns of photodetectors, thus each sensing surface has a corresponding set of columns of photodetectors. An evaluation unit determines the minimum reflectance or angle of resonance at each sensing surface.
U.S. Pat. No. 5,327,225 discloses an SPR sensor configuration in which a fiber optic is connected at one end to a laser, and at the other end to a detector. The fiber optic is coated with a metal layer and with an overlay or underlay material. The sensor can be used in biochemical and chemical applications when a test sample contacts the sensor. The overlay or underlay material can be like the sensitive layers of the above-mentioned disclosures, e.g. antibody or antigen layer. As in the above-mentioned disclosures, binding of antigen to antibody, for instance, changes the refractive index of the sample and thus the SPR signal.
U.S. Pat. No. 5,485,277 discloses an SPR sensor configuration which also can be used as a biosensor. It includes a metal film coated waveguide cartridge. The waveguide cartridge includes a planar waveguide coated with a metal film. The waveguide has a plurality of reflector surfaces within it. A sample flow cell is adjacent to the waveguide cartridge. A source of TM-polarized light is optically connected to the waveguide. A cylindrical diverging lens is optically connected to the waveguide. A plurality of photodetectors is optically connected to the cylindrical diverging lens. This configuration provides reflector surfaces within the waveguide and the SPR interface directly on the waveguide.
The SPR sensor of this invention can be, among others, a prism, waveguide, or light pipe. It can be single mode or multi-mode. It can be zero-order or first-order. U.S. provisional application Nos. 60/005,878, 60/007,027, 60/009,169 and corresponding U.S. applications (attorney docket nos. 89-95, 89A-95, and 101-95, respectively) filed Oct. 25, 1996, and incorporated in their entirety by reference herein disclose zero-order and first-order planar lightpipe sensors. These sensors can utilize various optical input and detection schemes.
If there are several different species present in solution at the surface of the sensor, the sensor will measure an effective index of refraction which is a function of all the species. In order to get around this lack of selectivity, many researchers have functionalized the surfaces of the SPR sensors, so that they selectively bind specific molecules (Jordan, C. E. et al. (1994), "Characterization of Poly-L-lysine adsorption onto alkanethiol-modified gold surfaces with polarization-modulation fourier transform infrared spectroscopy and surface plasmon resonance," Langmuir 10:3642-3648). This typically involves complicated and time-consuming chemistry, and can result in sensors that are stable over only a short period of time.
As an alternative to this approach, the present invention combines electrochemical methods and SPR to detect unknown species.